The Staphylococcus aureus cid and lrg operons encode a novel regulatory system that is important in the control bacterial death and lysis. Although fundamentally analogous to bacteriophage-encoded holins and antiholins that regulate the lysis of cells during the lytic cycle, the cid and lrg genes are thought to encode a widely conserved regulatory system that is fundamental to bacterial physiology. Recent studies have demonstrated that the biological function of this system is to control the cell death and lysis of a subpopulation of cells during biofilm development and that the released genomic DNA plays an important structural role as a component of the biofilm matrix. The objective of this proposal is to test the hypothesis that the proteins encoded by this system are the effectors of cell death and lysis and that their coordinate expression dictates the fate of the bacterial cells during biofilm development. This hypothesis will be tested in three specific aims. The first aim will characterize Cid and Lrg function using a combination of genetic and biochemical approaches. The second aim will utilize confocal microscopy and flow cytometry to explore the pattern and impact of cid/lrg expression during biofilm development. The third and final aim will study the developmental control of cid/lrg expression by focusing on the CidR transcriptional regulator and defining the molecular interactions that control cid and lrg promoter activity. The results of these studies will reveal new insight into the molecular mechanisms controlling cell death and lysis, as well as the control elements regulating these processes within a developing biofilm. They will also provide novel insight into the mechanisms involved in the development of antibiotic tolerance, thus potentially uncovering promising new antibiotic targets and innovative therapeutic strategies. Finally, the insight gained will serve as a paradigm for the elucidation of similar processes in other bacterial species. PUBLIC HEALTH RELEVANCE: Bacterial infections are a growing threat to human health around the world primarily due to their increasing ability to cause infections recalcitrant to antibiotic therapy. The research described in this proposal focuses on the novel concept that bacteria regulate their own cell death as part of complex developmental processes. The results generated by these studies will provide a better understanding of the molecular mechanisms controlling bacterial cell death and will lead to novel and more effective therapeutic strategies to combat infections.